Chemically self-assembled antibody nanorings (CSANs): design and characterization of an anti-CD3 IgM biomimetic.
Chemically self-assembled antibody nanorings (CSANs): design and characterization of an anti-CD3 IgM biomimetic.
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DOI：
10.1021/ja107153a
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发表时间：
2010-12-08
影响因子：
15
通讯作者：
Wagner CR
中科院分区：
文献类型：
--
作者：
Li Q;So CR;Fegan A;Cody V;Sarikaya M;Vallera DA;Wagner CR
已经开发出许多巧妙的重组方法来概括 IgG（二价）和 IgA（四价）的价态。尽管通过将单克隆抗体或单链抗体与纳米颗粒和脂质体缀合已经获得了更高的合成价，但尚未设计出一种制备重组抗体的方法，该重组抗体的价态与 IgM 相似（十价），但比抗体颗粒缀合后通常发现的价态要低得多。最近，我们开发了一种设计二价化学自组装抗体纳米环（CSAN）的方法。我们现在报道了由大肠杆菌 DHFR 二聚体和甲氨蝶呤二聚体 (MTX2-C9) 组成的纳米环亚基的晶体结构，该二聚体包含可见的九个亚甲基连接体，以及从该亚基制备 CSAN 的方案，其化合价类似于 IgM，范围为 8-10 个单链抗体 (scFv)。多价 CSAN 由融合蛋白二氢叶酸还原酶 (DHFR)-DHFR-antiCD3 scFv 可逆组装而成，在两个 DHFR 支架蛋白之间含有单个甘氨酸接头。我们还证明，与亲本二价抗 CD3 单克隆抗体 (MAB) 类似，抗 CD3 CSAN 选择性结合 CD3+ 白血病细胞，并通过需要胆固醇、肌动蛋白聚合和蛋白酪氨酸激酶激活的小窝蛋白独立途径进行快速内化。虽然用单克隆抗体治疗会导致 T 细胞激活，并且表面展示的 T 细胞受体 (TCR) 几乎完全丧失（即 90%），但用抗 CD3 CSAN 治疗外周血单核细胞 (PBMC) 后，只有 25-30% 的 TCR 下调，并且没有观察到显着的 T 细胞增殖。与增殖结果一致，在用多价或二价抗 CD3 CSAN 处理的 PBMC 表面上发现的 CD25（IL-2 受体）分别比用亲本 MAB 处理的 PBMC 少 15-25%。与源自 MAB 的 F(ab')2 的比较实验证实，MAB 对 T 细胞的激活依赖于 Fc 结构域，因此依赖于 PBMC T 细胞与辅助细胞（如巨噬细胞）的相互作用。总而言之，我们的结果表明，化合价范围为 2 至 8 的抗 CD3 CSAN 可用于将放射性核素、药物或潜在的寡核苷酸递送至 T 细胞，而不会像其他抗体偶联纳米颗粒所观察到的那样，产生 MAB 激活的有害影响。此外，CSAN构建体可适用于制备其他多价scFv。
A number of clever recombinant methodologies have been developed that recapitulate the valencies of IgG’s (bivalent) and IgA’s (tetravalent). Although, higher synthetic valencies have been achieved by conjugation of either monoclonal antibodies or single-chain antibodies to nanoparticles and liposomes, a method for the preparation of recombinant antibodies with valencies similar to IgM’s (decavalent) but considerably less than what is generally found after antibody particle conjugation has yet to be devised. Recently, we have developed a methodology for the design of bivalent Chemically Self-Assembled Antibody Nanorings (CSANs). We now report the crystal structure of the nanoring subunit composed of the E. coli DHFR dimer and a methotrexate dimerizer (MTX2-C9) containing visible nine methylene linker and a protocol for the preparation of CSANs from this subunit with valencies similar to IgMs, ranging from 8–10 single chain antibodies (scFvs). The multivalent CSANs were reversibly assembled from a fusion protein dihydrofolate reductase (DHFR)-DHFR-antiCD3 scFv containing a single glycine linker between the two DHFR scaffolding proteins. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (MAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells, and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e. 90%) of surface displayed T-cell receptor (TCR), only 25–30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15–25 % less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental MAB. Comparative experiments with F(ab')2 derived from the MAB confirm that the activation of the T-cells by the MAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious affects of activation observed for MAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.
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DOI：
10.1083/jcb.101.2.610
发表时间：
1985-08
期刊：
The Journal of cell biology
影响因子：
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作者：
Bronner-Fraser M
通讯作者：
Bronner-Fraser M
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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通讯作者：
Cowtan, K
影响因子：
4.4
作者：
Miller, K;Meng, G;Presta, L
通讯作者：
Presta, L
DOI：
10.1002/prot.10286
发表时间：
2003-02-15
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
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作者：
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通讯作者：
Richardson, DC
DOI：
10.1073/pnas.90.14.6444
发表时间：
1993-07-15
影响因子：
11.1
作者：
HOLLIGER, P;PROSPERO, T;WINTER, G
通讯作者：
WINTER, G